Five US studies using the Mg tolerance test, the ?gold standard? test of magnesium (Mg) status, indicated that >50% of participants had Mg deficiency. In our ongoing US trial, we have found a similar result. In growing recognition of the importance of Mg in human health, very recently, Mg was selected by the US Federal Dietary Reference Intake (DRI) Committee to update the DRI. However, current available human data are not conclusive. Further human studies are necessary to develop more accurate DRI and RDA for Mg. TNF (tumor necrosis factor)-induced necroptosis, a newly discovered pathway of regulated necrosis, is more inflammatory than apoptosis. Animal studies revealed very critical roles of necroptosis in inflammation and many human inflammatory diseases. Recently, TRPM7 (i.e. Transient Receptor Potential Melastatin 7, an ion channel essential to Mg absorption and homeostasis) was identified as a downstream effector of necroptosis. Consistent with in vitro data, Mg affected the growth of Bifidobacterium and Lactobacillus, two major beneficial bacteria in the gut, as well as systemic inflammation and metabolic disorders in mice. Our pilot findings from a randomized trial suggest reducing the Ca/Mg ratio through Mg supplementation may increase Bifidobacterium and Lactobacillus, accompanied by significant reductions in TRPM7 and COX-2 (cyclooxygenase 2) expression in rectal tissues. In addition to microbiota-host interaction on inflammatory response, many enzymes, including those involved in fermentation, transport and metabolism of carbohydrates, are dependent on the Ca/Mg ratio or Mg concentrations. In our pilot metagenomic study, we found all the significantly changed biologic functions within the microbial community caused by a reduction in the Ca/Mg ratio are biologically dependent on the Ca/Mg ratio or Mg concentrations. It is striking that the functions with significant changes in stool samples were centered on the fermentation of carbohydrates and energy metabolism while the functions in rectal swabs were related to immune response. Rectal tissues had a distinct functional profile. Our pilot data are highly novel and promising and have very broad clinical and public health significance for many inflammation-related diseases or metabolic disorders. However, due to the small sample size in our pilot study, these findings should be confirmed in larger studies which will also address many novel scientific inquiries based on our pilot data, such as other biomarkers in necroptosis-induced inflammation as well as microbial species and functions. In Aim 1, we will evaluate the effects of reducing the Ca/Mg ratio on colorectal microbiota abundance (i.e. Bifidobacterium and Lactobacillus measured by high-throughput quantitative PCR) and expression of biomarkers in necroptosis-related pathways in rectal tissue assayed by immunohistochemistry and serum TNF-? in the ?Personalized Prevention of Colorectal Cancer Trial [PPCCT, R01CA149633; PI, Dai & Yu]? comparing Mg treatment (12 weeks) vs. placebo (n=240). In Aim 2, we will evaluate the effects of reducing the Ca/Mg ratio on colorectal microbiota function by a two-phase study.